ABSTRACT A high protein diet has been shown in preclinical rodent models and clinical trials to be an effective obesity treatment that is associated with greater loss of body weight and fat mass and increased satiety compared to isocaloric standard protein diets. However, the mechanisms of this response have not been fully elucidated. I recently demonstrated in a rodent model that a high protein diet induces shifts in the intestinal microbiome including a bloom of Akkermansia muciniphila, a microbe reported to have an anti-obesity effect. Based on these preliminary studies, I hypothesize that a high protein diet induces alterations in the intestinal microbiome that mediate its clinical efficacy for obesity. To test this, I will study 216 overweight and obese Veterans (BMI 27-40) who will be randomized 1:1 to isocaloric high protein (30%) or normal protein (15%) 1500 calorie diets for 16 weeks utilizing existing clinical infrastructure at the West Los Angeles VA Medical Center established for a recently completed clinical trial of a high protein diet. In Aim 1, the effect of a high protein diet on the composition and function of the intestinal microbiome will be assessed by 16S rRNA sequencing, shotgun metagenomics, and metabolomics. I predict that following a high protein diet, there will be a major shift in the composition of the intestinal microbiome to favor colonization with Akkermansia and other microbes that promote fat loss compared to the normal protein diet. I anticipate that this change in microbial composition will be associated with alterations in the metagenome ? including reduced abundance of bacterial genes involved in carbohydrate utilization ? and the metabolome. A total of 11 fecal samples will be collected during the study period to determine the kinetics of microbial changes. In Aim 2, fecal microbial, metagenomic, and metabolomic predictors of response to a high protein diet will be identified. Response will be measured by weight loss, reduced body fat, decreased hepatic steatosis, altered lipid profiles, reduced hemoglobin A1c, decreased high sensitivity C-reactive protein, and increased satiety. Predictors will be generated using the baseline microbiome data as well as samples collected at each time point during treatment. I will also evaluate whether specific microbes, genes, and metabolites are associated with circulating levels of hormones affecting satiety (leptin, ghrelin glucagon, glucagon-like peptide-1, peptide YY, and pancreatic polypeptide). In Aim 3, germ-free mice will be colonized with fresh frozen feces obtained at baseline or after 16 weeks of dietary intervention to establish a causal relationship between alteration of the microbiome on a high protein diet and susceptibility to obesity. These humanized gnotobiotic mice will be placed on a Western diet to induce obesity and compared for weight gain, body fat accumulation, insulin sensitivity, and satiety/hunger hormone levels. In parallel, mice with pre-existing diet-induced obesity will be colonized with post-dietary intervention human microbiota and placed on a standard diet. Weight loss, body fat reduction, insulin sensitivity, and hormone levels will be compared between the two groups. The results of the proposed study will provide insight into the specific microbes that drive the clinical response to a high protein diet and may identify candidate anti-obesity microbes that could be further developed into novel microbial therapeutics.